Biologically pure strain of Enterococcus falcium FERM BP-4504

ABSTRACT

Disclosed is an alkali-treated bagasse prepared by softening a bagasse with calcium oxide together with or without sodium hydroxide while preventing the substantial decomposition of cellulose and hemicellulose, a bagasse feed and a fermented bagasse feed prepared from the alkali-treated bagasse, and their preparations and uses as well as bacteria for fermenting the alkali-treated bagasse. These bagasse and bagasse feeds save sugar-refinery industries from the difficulties to treat bagasse deemed as an unutilized industrial waste, and livestock farming industries from the shortage of roughage, and further is extremely significant in agriculture, industries of sweetening products, feed industries and livestock product processors. A bacterium strain particularly useful for the fermentation is Enterococcus faecium FERM BP-4504.

This is a continuation of parent application Ser. No. 08/364,489 filedDec. 27, 1994, now U.S. Pat. No. 5,548,418.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an alkali-treated bagasse and itspreparation and uses, more particularly to an alkali-treated bagassewhose structure is softened while preventing the decomposition ofcellulose and hemicellulose, and a bagasse feed and a fermented bagassefeed prepared from said alkali-treated bagasse, as well as to theirpreparation and uses, and to a microorganism utilizable for fermentingsaid alkali-treated bagasse.

2. Description of the Prior Art

Although having been utilized in part as a fuel source, bagasse is asqueezed waste of sugar cane which contains a vast of cellulose andhemicellulose for which there have not yet been found other usefulapplications. The problem is that more than 100 million tons of bagasseproduced annually worldwide have been still unutilized.

On the other hand, livestock fed with grass, or ruminants like cattleand sheep assimilate cellulose and hemicellulose of plants in nature,differerently from monogastric animals like human beings, pigs andpoultry, and further physiologically utilize said cellulose andhemicellulose as an energy source.

Recently, the consumption of livestock products such as beef and dairyproducts has been increased so that an intensive livestock farmingsystem has been remarkably developed to breed a lot of cattle standingin the same direction together in a small shed, and said intensivelivestock farming system has tightened the supply of feed grass requiredfor breeding and also has led the rapid increase of the demand forroughage containing cellulose and hemicellulose substitutable for feedgrass.

There have been many studies to utilize bagasse as roughage for a longtime, however, in addition of cellulose and hemicellulose, bagassecontains a considerable amount of lignin having a tight linkage withfibre like cellulose and said linkage tightens the structure of bagassein the same manner as bamboo. While ruminants can assimilate bagasseitself, the digestibility of bagasse for ruminants is relatively low andthe taste and nutrient value of bagasse for ruminants is veryunpreferable. It is known that, when cattle assimilate a raw bagasse,there is a danger that pieces of said bagasse could be stuck into thewalls of cattle's rumen.

For improving a nutrient value of bagasse, there were many proposals ofincreasing the digestibility of bagasse by decomposing lignin to softenthe structure of bagasse. Most of said proposals comprise treatingbagasse with an alkaline reagent and succeedingly fermenting theresultant alkali-treated bagasse. In such alkali treatment, as describedin Biotechnology and Bioengineering, volume 26, pp. 426-433 (1984), itis known to use as an alkaline reagent sodium hydroxide and calciumhydroxide as well as sodium carbonate. In the fermentation of analkali-treated bagasse, for instance, as described in Animal FeedScience and Technology, volume 9, pp. 1-17 (1983), an ensilage of analkali-treated bagasse with sodium hydroxide is known.

We eagerly studied these conventional methods and found that in the caseof an alkali treatment with sodium hydroxide, because of its strongalkalinity, a relatively small amount of sodium hydroxide was requiredto raise the pH of bagasse mixtures to a relatively-high level so thatlignin was readily decomposed to soften the structure of bagasse withoutreadily decomposing cellulose and hemicellulose necessary for roughage.In addition, it was found that the pH level of alkali-treated bagassewas decreased, specifically, said pH level was decreased very graduallyover a long period of time to the range wherein lactic acid bacteriawere proliferative, and further that a long period of 25 to 90 days wasrequired for preparing a fermented bagasse feed from said alkali-treatedbagasse. It was found that, in order to avoid said disadvantage and toincrease rapidly the pH level of alkali-treated bagasse, saidalkali-treated bagasse should be neutralized with an acid solution andthere were many other drawbacks in conventional methods.

It was found that in the case of using calcium hydroxide and sodiumcarbonate, because of their relatively weak alkalinity, the amounts ofalkaline reagents used was increased to the level of about 12 to 30 w/w% of the bagasse, d.s.b. (the wording of "w/w %" as referred to theinvention will be abbreviated as "%" hereinafter), the cost of alkalitreatment was raised and ruminants consuming excessively alkalinereagents desired a large amount of water so that they excreted a largeamount of urine. Therefore it was found that the conventional alkalitreatment has an extreme drawback by adversely physiologically affectingruminants.

SUMMARY OF THE INVENTION

The present invention provides an alkali-treated bagasse which overcomesthe above drawbacks and its uses, and considering a vast of demands forsaid bagasse, the present invention provides a preparation of saidbagasse readily at a relatively-low cost and in a relatively-shortperiod.

We, in order to overcome the above object, have studied eagerly the useof to an alkaline reagent in treatment of bagasse. As a result, weunexpectedly found that calcium oxide which has not been consideredconventionally is suitable for the production of an alkali-treatedbagasse and also preferable for the preparation of a fermented bagassefeed from said alkali-treated bagasse, and accomplished the presentinvention.

Furthermore, we found that using as alkaline reagents sodium hydroxidetogether with calcium oxide is far more suitable than and in no wayinferior to using only calcium oxide and further is preferable for thepreparation of an alkali-treated bagasse as well as a fermented bagassefeed using said alkali-treated bagasse, and accomplished the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention utilizing calcium oxide together with or withoutsodium hydroxide as alkaline reagents for an alkali-treatment of bagassehas the following characteristics (1) producing an alkali-treatedbagasse in which the structure is softened without almost decomposingcellulose and hemicellulose as an effective component of bagasse; (2)attaining the object of an alkali-treatment by using a relatively-smallamount of an alkaline reagent in a relatively-short period becausecalcium oxide is an active alkaline reagent or absorbs water and isexothermic enough to promote alkali reaction; (3) inoculating a seedculture of a lactic acid bacterium in an early stage withoutneutralization by acid because the pH level of an alkali-treated bagasseis decreased relatively rapidly; (4) suitable for a large-scaleproduction of an alkali-treated bagasse and a fermented bagasse feedusing said alkali-treated bagasse because of easily controllable,short-period and relatively low cost production; (5) preferablenutritionally for ruminants because a relatively-small amount ofalkaline reagents relative to bagasse is used without considering theexcess intake of alkaline reagents and because the nutritive value ofbagasse is intensified by adding thereto an appropriate amount ofcalcium or minerals; and further (6) a fermented bagasse feed obtainableby the present invention has a relatively-high quality and excellentdigestibility and good taste, and (7) an alkali-treated bagasse obtainedand a bagasse feed as well as a fermented bagasse feed prepared fromsaid alkali-treated bagasse is usable preferably as a livestock feed aswell as an organic fertilizer.

The present invention is explained in detail as follows:

A bagasse usable in the present invention includes those having a watercontent of about 40 to 50% as produced in sugar refineries. However, ifnecessary, a dried and stored bagasse is usable. A preferable watercontent for use in an alkali-treatment is in the range from the lowerlimit wherein both bagasse and alkaline reagents could be mixedsufficiently to the upper limit wherein an alkali solution is preventedfrom leaking out of an alkali-treated bagasse. Specifically, a watercontent of about 40 to 90%, although that of about 45 to 80% is moresuitable.

As to the calcium oxide usable in the present invention, those having ahigher activity, namely, those as much water as possible are desirable,which can be in the form of granules or powder, if necessary. The amountof calcium oxide suitably used in the invention is in the range of notexceeding 5% with respect to bagasse, preferably, in the range of 2 to4%, on a dry solid basis (d.s.b.). The amount of sodium hydroxidesuitably used in the invention is in the range of not exceeding theamount of the calcium oxide, preferably, not more than 3%.

In the case of adding alkaline reagents to a bagasse under thecoexistence of water, any method of mixing alkaline reagents with thebagasse containing a certain amount of water can be favorably employed,for example, at first, bagasse is macerated and succeedingly mixed withalkaline reagents, or vice versa ad libitum. These procedures can beconducted at an ambient temperature, usually, a temperature in the rangeof about 10° to 35° C.

The initial pH level of the bagasse thus obtained is usually not lessthan about pH 10.3, favorably, in the range of about pH 10.5 to 12, andby allowing said bagasse to stand at an ambient temperature, analkali-treated bagasse having not more than pH 10, preferably, not morethan pH 9.5 is readily obtainable, and thus the object ofalkali-treatment is attained in a short time of about 10 to 40 hours.

The alkali-treated bagasse thus obtained is softened withoutsubstantially damaging cellulose and hemicellulose, particularly, in thecase of using calcium oxide together with sodium hydroxide, incomparison of the single use of calcium oxide, the lignin of thealkali-treated bagasse is decomposed to soften further its structurewithout substantially damaging cellulose and hemicellulose, and is asuitable raw material of a fermented bagasse feed. The alkali-treatedbagasse can be advantageously used, without fermentation, as variousfeed by mixing it with such nutrients as an energy source like molassesand organic acid and protein like a de-fatted soybean and a cottonseedlees.

We found that the alkali-treated bagasse or a bagasse feed obtainable byblending said alkali-treated bagasse with various nutrients is suitableas an organic fertilizer which is appropriately decomposed in soil andexhibits remarkably its ability of providing a nutrient supplement tosoil and of improving conditions as well as humidity of soil. In casethat the alkali-treated bagasse or the bagasse feed is utilized as afertilizer, a relatively-small amount of the bagasse or the feed,usually acres selected from the range of about 40 to 500 kg in 10, canbe scattered in soil, which is variable depending on the composition ofthe bagasse or the feed, conditions of soil and the kind of crops to begrown.

For the purpose of preparing a fermented bagasse feed from analkali-treated bagasse, the alkali-treated bagasse is fermentednaturally, preferably, by adding a seed culture of lactic acid bacteriaand nutrients to the alkali-treated bagasse and then fermenting themixture after or immediately before the pH level of the alkali-treatedbagasse decreases to a pH range wherein a seed culture of lactic acidbacteria proliferates. In such a case, if necessary, an alkali-treatedbagasse can be partially neutralized with an acid solution to meet theminimum pH level for the proliferation of lactic acid bacteriainoculated.

The lactic acid bacteria usable in the present invention include thosecapable of fermenting in a medium containing an alkali-treated bagasse,for instance, those of the genera Enterococcus, Lactobacillus,Streptococcus and Pediococcus are preferable. Specifically, Enterococcusfaecium HL-5 (FERM BP-4504) belonging to the genus Enterococcus newlyisolated by the present inventors, which proliferates in alkalisolutions of not less than pH 9.5 or in high concentration salinesolutions containing 6.5% sodium chloride and further produce lacticacid to decrease the pH of the alkali-treated bagasse, is suitable forpreparing a fermented bagasse which is preferred.

We found that the present microorganism is capable of proliferating inalkaline nutrient media of pH not more than 10 containing analkali-treated bagasse, desirably, those having a pH not less than 9 toa pH not more than 10, more desirably, not less than 9.5 to a pH notmore than 10, and said microorganism can be seeded at a relatively earlystage wherein the pH decrease of alkali-treated bagasse is close to pH10, in other words, a desirable alkali treatment period can be usually 1to 2 days or less. Furthermore the microorganism is characterized inthat it satisfactorily shortens the preparation period of a fermentedbagasse feed to about 3 to 6 days. When inoculated into the abovealkali-treated bagasse with calcium oxide whose pH level readilydecreases, the microorganism advantageously shortens the productionperiod of fermented bagasse feeds.

The results of identification study of the microorganism, the generaEnterococcus HL-5 newly isolated from ensiled corns by the presentinventors, are shown as follows. The identification study was conductedaccording to Biseibutsu no Bunrui to Doutei (Classification andIdentification of Microorganism)(Edited by Hasegawa, GakujyutsuShutsupan Center, 1985).

[A. Morphology]

(1) Characteristics of cells when incubated at 37° C. in MRS agar

Usually existing in a coccus form of 0.9 to 1.2 μm;

Existing in a coupled- or short linked-form;

Motility: Negative;

Asporogenicity; and

Gram stain: Positive.

(2) Characteristics of cells when incubated at 37° C. in PG agar

Usually existing in a coccus form of 0.9 to 1.2 μm;

Existing in a coupled- or short linked-form;

Motility: Negative

Asporogenicity; and

Gram stain: Positive.

[B. Cultural properties]

(1) Characteristics of colony formed when incubated at 37° C. in MRSagar plate

Shape: Circular colony having a diameter of about 1 to 2 mm after 3 daysincubation;

Rim: Entire;

Projection: Hemispherical shape;

Gloss: Wet-look gloss;

Surface: Smooth; and

Color: Milk white and semitransparent.

(2) Not liquefying MRS gelatin when stab cultured at 37° C.

(3) Forming acid and gelatinizing litmus milk when incubated therein at37° C.

[C. Physiological properties]

(1) Catalase: Negative

(2) Oxidase: Negative

(3) Liquefaction of gelatin: Negative

(4) Hydrolysis of casein: Negative

(5) Hydrolysis of arginine: Positive

(6) Tolerance to 40% bile: Positive

(7) Hemolysis: Negative

(8) Hydrolysis of hippurate: Positive

(9) Hydrolysis of aesculin: Positive

(10) Growth at 10° to 45° C.: Positive

(11) Growth at pH 9.6: Positive

(12) Growth in 6.5% NaCl: Positive

(13) Oxygen requirements: Facultative an anaerobic

(14) Acid formation from carbon source

Amygdalin: Positive

Arabinose: Positive

Cellobiose: Positive

Aesculin: Positive

Fructose: Positive

Galactose: Positive

Glucose: Positive

Gluconic acid: Positive

Lactose: Positive

Maltose: Positive

Mannitol: Positive

Mannose: Positive

Melezitose: Negative

Melibiose: Positive

Raffinose: Positive

Rhamnose: Positive

Ribose: Positive

Salicin: Positive

Sorbitol: Negative

Sucrose: Positive

Trehalose: Positive

Xylose: Negative

Arbutin: Positive

Sorbose: Negative

(15) Main diamino acids of cell walls: Lysine

(16) Mol % glycine (G) plus cytosine (C): 38.6%

The above bacteriological properties were compared with those of knownstrains with reference to Bergey's Manual of Systematic Bacteriology,volume 2 (1986). As a result, based on the above properties, it wasrevealed that the microorganism was identified with a microorganism ofthe species of Enterococcus faecium except for the property that acidformation from sorbose was negative.

On the basis of the above result, the present inventors designated themicroorganism as a novel microorganism Enterococcus faecium HL-5 and, onthe date of Dec. 17, 1993, deposited it in the National Institute ofBioscience and Human-Technology Agency of Industrial Science andTechnology placed at 1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken, andit was accepted under the deposit number of FERM BP-4504. In the presentinvention, in addition to the above microorganism, the other strainsbelonging to the genus Enterococcus, which are capable of proliferatingin an alkali medium having a pH not less than 9.5 and containing analkali-treated bagasse, and also their mutants are utilizablepreferably. The other strains belonging to the genus Enterococcus usablein the present invention include, for instance, Enterococcuscasseliflavus IFO 3531, Enterococcus durans IFO 13131, Enterococcusfaecalis IFO 3791, Enterococcus faecium IFO 3535 and Enterococcus hiraeIFO 3181T.

It was found that, similarly to Enterococcus faecium HL-5 isolated bythe present inventors, lactic acid bacteria belonging to the generaEnterococcus are capable of proliferating in a relatively early stage ofthe pH decrease of alkali-treated bagasse, specifically, not less thanpH 9, desirably, capable of proliferating sufficiently in the stage ofnot less than pH 9.5, and realize an early stage inoculation, andtherefore the lactic acid bacteria are suitable for shortening thefermentation period.

The combination use of a lactic acid bacterium belonging to the genusEnterococcus and one or more members selected from lactic acid bacteriabelonging to the genera Lactobacillus, Pediococcus and Streptococcuswhich are capable of proliferating at a pH of less than and closer to 8is extremely desirable for the production of a high-quality fermentedbagasse feed having an excellent feed taste wherein the productionperiod is shortened.

The strains belonging to the genera Lactobacillus include, for instance,Lactobacillus rhamnosus IFO 3532, Lactobacillus plantarum IFO 3070,Lactobacillus sake IFO 3541, Lactobacillus acidophilus IFO 13952,Lactobacillus helveticus IFO 3809 and Lactobacillus brevis IFO 3345, andthe combination use of one or more members of said strains are suitablefor improving the preference of fermented bagasse feeds.

Furthermore, microorganisms belonging to the genera Pediococcus andStreptococcus, for instance, Pediococcus acid ilactici IFO 3076 andStreptococcus bovis IFO 12057 are utilizable advantageously.

An alkali-treated bagasse can be fermented by incorporating a seedculture of lactic acid bacteria and nutrients into the alkali-treatedbagasse, usually, by adding nutrients, if necessary, together with anappropriate amount of water to said alkali-treated bagasse, inoculatinga seed culture of lactic acid bacteria and then fermenting the mixtureat an ambient temperature, desirably, in the range of about 15° to 50°C., for instance, such a fermentation can be effected by fermentinganaerobically for about 2 or 4 days the above mixture contained in aflexible bag or lapped.

The nutrients usable in the invention include nutrients useful for theproliferation of lactic acid bacteria and/or for animals assimilatingthe fermented bagasse feed obtainable by the present invention, usually,one or more members of the group consisting of energy sources, protein,minerals and vitamins can be used. Specifically, for instance, molasses,sugar, dextrose, starch, organic acids, alcohols, press-ground grain,sorghum, barley, barley, pulverized rice, rice bran, wheat flour, wheatflour bran, corn, corn gluten, feed in general, de-fatted soybean,soybean casein, cottonseeds, cottonseed lees, rape seed lees, beer lees,milk, de-fatted dry milk, milk casein, milk serum, blood meal, bonemeal, feather meal, fish meal, urea, ammonium salts, calcium salts,magnesium salts, sodium salts, phosphates, iron salts, copper salts,zinc salts, water-soluble vitamins and lipid-soluble vitamins are usedarbitrarily.

Preferably, the above nutrients are incorporated usually in the range ofnot exceeding the amount of used bagasse, d.s.b. The wording of"incorporating an appropriate amount of water" as referred to thepresent invention means to incorporate the water into the contents inthe range from the lower limit proceeding lactic acid fermentation tothe upper limit preventing nutrient solutions from leaking out of theresultant fermented bagasse feed, usually in the range of about 40 to90%, desirably about 45 to 80%, and in the case of putting priority onpreservation of the fermented bagasse feed, the water content of about45 to 60% is suitable. The kind and amount of nutrients used in thefermentation can be selected arbitrarily depending on the finalproducts, for instance, roughage or well-balanced feed containingnutrients proportionately. The fermented bagasse feeds thus obtained areacidified by lactic acid and are excellent in preservation and taste,although these properties are variable dependently on the kind andamount of used nutrients. If necessary, it is feasible advantageouslythat in order to improve the preservation of said fermented bagassefeeds, they are dried by air or hot air to give a water content of notmore than 40%, preferably, to decrease said water content to not morethan 30% and then the fermented bagasse feeds dried thus are readilypreserved and utilized.

The fermented bagasse feeds thus obtained in the present invention arehigh-quality feeds having good taste and excellent digestibility. Thefermented bagasse feeds are utilizable mainly as a feed for ruminants,and if necessary, for monogastric animals like pigs and poultry,specifically said bagasse feeds are usable as a feed which is capable ofcontrolling and improving intestinal conditions and of preventinginfections. Usually cattle assimilate the present fermented feed ofabout 4 to 10 kg or more in a day, and the cattle increase their bodyweights by not less than about 2 kg/body in a day, preferably, not lessthan about 2.5 kg/body, depending on the kind of fermented bagasse feedsand the ages of the cattle.

It is feasible advantageously that, depending on the kind of animals tobe fed and their ages, a fermented bagasse feed can be arbitrarily mixedother nutrients.

We found that the bagasse feed is usable preferably as organicfertilizer appropriately decomposed in soil and exhibits remarkably itsability of providing a nutrient supplement to soil and of improvingconditions as well as humidity of soil. In case that the bagasse feed isutilized as a fertilizer, a relatively-small amount of the bagasse feed,usually selected from the range of about 60 to 800 kg in 10 acres, canbe scattered in soil, which is variable depending on the composition ofthe bagasse or the feed, conditions of soil and the kind of crops to begrown.

The following experiments explain the present alkali treatment usingcalcium oxide in detail:

Experiment 1

Effect of alkaline reagent on the preparation of alkali-treated bagasse

Bagasse was macerated with water to give a water content of 70% andmixed with as an alkaline reagent 3, 5 or 7% sodium hydroxide (NaOH) tobagasse, d.s.b., respectively and 2, 3, 5, or 7% calcium oxide (CaO) tobagasse, d.s.b., respectively as uniformly as possible, and the mixturewas allowed to stand at ambient temperature. The alkali-treated bagassethus obtained was analyzed for its changes of pH and components as wellas its structural flexibility.

The pH level of the alkali-treated bagasse was determined immediatelyafter the mixing and at 24 and 48 hours termination. The samples wereanalyzed by admixing 1 part by weight of an alkali-treated bagassespecimen with 2 parts by weight of refined water, allowing the mixtureto stand for 10 minutes, filtering the mixture, and measuring the pH ofthe resultant filtrated on a pH meter. As to components, raw bagasse andthe alkali-treated bagasse collected at 24 and 48 hours termination wererespectively divided into 8 samples, and their contents ofhemicellulose, cellulose and lignin were determined and their averagevalue calculated.

The above measurement was conducted in accordance with the Van Soest'sdetergent filter method as described in the 3.1 chapter of "DietaryFiber", pp. 38 to 46 (1982), published by Dai-ichi Shutupan Co., Ltd.,Tokyo, Japan. The components of raw bagasse essentially consisted of28.7% hemicellulose, 52.6% cellulose and 11.9% lignin, d.s.b.

The structural flexibility of the alkali-treated bagasse was analyzed bystudying its touch as clutched by hands wearing thin rubber gloves. Theresults were tabulated in Table 1.

In Table 1, numerical values pertaining to the item of hemicellulose,cellulose and lignin are designated as a ratio of the remaininghemicellulose, cellulose and lignin after alkali treatment as comparedto their initial contents in raw bagasse respectively.

As shown in Table 1, sodium hydroxide used conventionally tends todecrease the pH slowly in comparison with calcium oxide. As to thechange of components of bagasse, it was revealed that cellulose andhemicellulose important for roughage are remarkably decomposed andspoiled by sodium hydroxide treatment.

On the contrary, it was found that the present alkali-treated bagassewith calcium oxide, free of substantial decomposition of cellulose andhemicellulose, is advantageously used for a raw material of feeds.

Furthermore the alkali-treated bagasse with calcium oxide, whichdecreases its pH level relatively rapidly to the range wherein lacticacid bacteria for the production of fermented bagasse feeds proliferatedand making it possible to inoculate into the bagasse lactic acidbacteria at a relatively early stage, is preferably suitable for a rawmaterial of fermented bagasse feeds.

Experiment 2

Effect of the combination of calcium oxide and sodium hydroxide onalkali-treated bagasse

In accordance with the method of Experiment 1, bagasse was macerated togive a water content of 70%, the macerated

                                      TABLE 1                                     __________________________________________________________________________           Time (hr)                                                                     0  24              48                                                         pH pH HC C  L  F   pH HC C  L  F                                       __________________________________________________________________________    Control                                                                       Alkali                                                                            (%)                                                                       NaOH                                                                              3  10.8                                                                             10.3                                                                             79.4                                                                             93.5                                                                             75.3                                                                             Good                                                                              10.0                                                                             72.3                                                                             91.5                                                                             74.5                                                                             Good                                        5  11.3                                                                             10.7                                                                             65.7                                                                             88.2                                                                             72.8                                                                             Good                                                                              10.3                                                                             59.8                                                                             85.4                                                                             71.4                                                                             Good                                        7  12.4                                                                             11.5                                                                             53.6                                                                             81.3                                                                             66.4                                                                             Good                                                                              11.0                                                                             37.7                                                                             78.6                                                                             54.6                                                                             Good                                    The present invention                                                         CaO 2  10.4                                                                             8.9                                                                              99.2                                                                             99.3                                                                             82.2                                                                             Good                                                                              8.5                                                                              99.1                                                                             99.2                                                                             80.2                                                                             Good                                        3  10.7                                                                             9.3                                                                              98.1                                                                             99.5                                                                             80.6                                                                             Good                                                                              8.7                                                                              98.0                                                                             98.1                                                                             79.0                                                                             Good                                        5  11.2                                                                             10.4                                                                             97.4                                                                             97.4                                                                             77.5                                                                             Good                                                                              10.0                                                                             96.1                                                                             97.3                                                                             75.6                                                                             Good                                        7  11.9                                                                             11.2                                                                             91.6                                                                             94.8                                                                             75.2                                                                             Good                                                                              10.5                                                                             90.3                                                                             93.6                                                                             74.4                                                                             Good                                    __________________________________________________________________________     HC: hemicellulose, C: cellullose, L: lignin, and F: flexibility               *Numerical values (%) pertaining to hemicellulose, cellulose and lignin       mean the ratios of the remaing hemicellulose, cellulose and lignin as         compared to their initial contents in raw bagasse.                       

bagasse was mixed 2 or 3% calcium oxide (CaO), and 1, 2 or 4% sodiumhydroxide (NaOH) with respect to bagasse, d.s.b., respectively, and thealkali-treated bagasse thus obtained was studied for its changes of pHand components as well as its structural flexibility. The results weretabulated in Table 2.

As shown in Table 2, it was unexpectedly found that the use of acombination of calcium oxide and sodium hydroxide as alkaline reagentsprovided the similar advantageous result as Experiment 1 using onlycalcium oxide. In other words, each sample of alkali-treated bagasse inExperiment 2 was sufficiently flexible without decomposing cellulose andhemicellulose similarly as in the case of using only calcium oxide, andit was revealed that the use of a combination of calcium oxide andsodium hydroxide is advantageous.

Specifically, it was revealed that incorporating sodium hydroxide in therange of not exceeding the amount of calcium oxide used allows theeffective components of cellulose and hemicellulose in a bagasse toremain sufficiently intact and also to decompose, lignin and the bagassethus alkali-treated is flexible sufficiently and suitable for analkali-treated bagasse.

The following examples A illustrate the preparations of analkali-treated bagasse. Examples B illustrate the uses of a fermentedbagasse feed in detail, but are by no means limitative of the presentinvention:

Example A-1

Bagasse was macerated to give a water content of 70% and the resultantmacerated bagasse was added with 4% calcium oxide to bagasse, d.s.b., asalkaline reagents and

                                      TABLE 2                                     __________________________________________________________________________            Time (hr)                                                             Alkali (%)                                                                            0  24              48                                                 CaO NaOH                                                                              pH pH HC C  L  F   pH HC C  L  F                                      __________________________________________________________________________    2   1   10.8                                                                             9.4                                                                              94.4                                                                             99.5                                                                             79.7                                                                             Good                                                                              9.0                                                                              98.5                                                                             99.1                                                                             78.3                                                                             Good                                   2   2   11.1                                                                             10.3                                                                             99.1                                                                             99.3                                                                             77.0                                                                             Good                                                                              9.6                                                                              98.2                                                                             98.4                                                                             75.0                                                                             Good                                   2   4   12.0                                                                             11.3                                                                             78.6                                                                             95.4                                                                             73.4                                                                             Good                                                                              10.4                                                                             76.6                                                                             93.5                                                                             70.2                                                                             Good                                   3   1   11.0                                                                             10.1                                                                             98.5                                                                             99.3                                                                             77.4                                                                             Good                                                                              9.1                                                                              97.2                                                                             99.2                                                                             76.2                                                                             Good                                   3   2   11.6                                                                             10.4                                                                             98.2                                                                             98.1                                                                             75.0                                                                             Good                                                                              9.7                                                                              97.1                                                                             99.1                                                                             74.3                                                                             Good                                   3   4   12.0                                                                             11.3                                                                             74.6                                                                             92.4                                                                             72.2                                                                             Good                                                                              10.4                                                                             72.5                                                                             90.2                                                                             68.7                                                                             Good                                   __________________________________________________________________________     HC: hemicellulose, C: cellullose, L: lignin, and F: flexibility               *Numerical values (%) pertaining to hemicellulose, cellulose and lignin       mean the ratios of the remaing hemicellulose, cellulose and lignin as         compared to their initial contents in raw bagasse.                       

allowed to stand overnight to obtain an alkali-treated bagasse having pHabout 9.7.

The present bagasse is usable as a raw material for fermented bagassefeeds or preferably utilizable without fermentation as a roughage forformula feeds.

Example A-2

Bagasse was macerated to give a water content of 60%, and the resultantmacerated bagasse was mixed with as alkaline reagents 3% calcium oxideand 1% sodium hydroxide to bagasse, d.s.b., respectively, and themixture was allowed to stand overnight to obtain an alkali-treatedbagasse having pH about 10.2.

Similarly as the product in Example A-1, the product is usable as a rawmaterial for fermented bagasse feeds or preferably utilizable as aroughage for formula feeds.

Example A-3

Bagasse having a water content of about 47% was mixed with as alkalinereagents 3% calcium oxide and 2% sodium hydroxide to bagasse, d.s.b.,respectively, and the mixture was allowed to stand overnight to obtainan alkali-treated bagasse having about pH 9.6.

Similarly as the product in Example A-1, the product is usable as a rawmaterial for fermented bagasse feeds or preferably utilizable as aroughage for formula feeds.

Example A-4

Bagasse having a water content of about 45% was mixed with as alkalinereagents 2% calcium oxide and 2% sodium hydroxide to bagasse, d.s.b.,respectively, and the mixture was allowed to stand overnight to obtainan alkali-treated feed bagasse having pH about 10.3.

Similarly as in Example A-1, the product is usable as a raw material forfermented bagasse feeds or preferably utilizable as a roughage forformula feeds.

Example B-1

A seed culture of Enterococcus faecium FERM BP-4504 was inoculated intoa nutrient culture medium containing 100 parts by weight of analkali-treated bagasse obtained by the method in Example A-1, 10 partsby weight of molasses, 0.2 parts by weight of urea and 0.2 parts byweight of salt as nutrients, and the mixture was covered with a plasticsheet and fermented at a room temperature for 2 days to obtain afermented bagasse feed.

The fermented bagasse feed, sufficiently digestible, is preferablysuitable as a high-quality feed having an excellent taste for ruminants.It is advantageously feasible to increase the feed value of thefermented bagasse feed and to produce a feed for monogastric animalslike pigs and poultry by blending other nutrients with the fermentedbagasse feed.

Example B-2

Seed cultures of Enterococcus casseliflavus IFO 3531 and Lactobacillusplantarum IFO 3070 were inoculated into a nutrient culture mediumcontaining 100 parts by weight of an alkali-treated bagasse obtained bythe method in Example A-2 and as nutrients 20 parts by weight of wheatflour bran, 10 parts by weight of molasses, 0.2 parts by weight ofammonium phosphates and 40 parts by weight of water, the mixture wasfermented similarly as in Example B-1 to obtain a fermented bagassefeed.

The fermented bagasse feed, sufficiently digestible, is preferablysuitable as a high-quality feed having an excellent taste for ruminants.It is advantageously feasible to increase the feed value of thefermented bagasse feed and to produce a feed for monogastric animalslike pigs and poultry by blending other nutrients with the fermentedbagasse feed.

Example B-3

Seed cultures of Enterococcus faecalis IFO 3791 and Lactobacillus brevisIFO 3345 were inoculated into a nutrient culture medium containing 100parts by weight of an alkali-treated bagasse obtained by the method inExample A-3 and as nutrients 10 parts by weight of wheat flour bran, 10parts by weight of de-fatted soybean, 5 parts by weight of corn meal, 10parts by weight of molasses, 0.2 parts by weight of ammonium phosphateand 60 parts by weight of water, the mixture was fermented in a flexiblebag at a room temperature for 2 days to obtain a fermented bagasse feed.

The fermented bagasse feed, sufficiently digestible, is preferablysuitable as a high-quality feed having an excellent taste for ruminants.It is advantageously feasible to increase the feed value of thefermented bagasse feed and to produce a feed for monogastric animalslike pigs and poultry by blending other nutrients with the fermentedbagasse feed.

Example B-4

Seed cultures of Enterococcus faecium FERM BP-4504, Lactobacillusrhamnosus IFO 3532 and Lactobacillus acidophilus IFO 13952 wereinoculated into a nutrient culture medium containing 100 parts by weightof an alkali-treated bagasse obtained by the method in Example A-4 andas nutrients 20 parts by weight of wheat flour bran, 5 parts by weightof press-ground barley, 5 parts by weight of rice bran, 10 parts byweight of molasses, 5 parts by weight of milk serum and 70 parts byweight of water, the mixture was fermented in a flexible bag at a roomtemperature for 3 days to obtain a fermented bagasse feed.

The fermented bagasse feed, sufficiently digestible, is preferablysuitable as a high-quality feed having an excellent taste for ruminants.It is advantageously feasible to increase the feed value of thefermented bagasse feed to produce a feed for monogastric animals likepigs and poultry by blending other nutrients with the fermented bagassefeed.

Example B-5

A fermented and dried bagasse feed was prepared by drying in hot air afermented bagasse feed prepared by the method in Example B-3 to obtain adried and fermented bagasse feed having a water content of 20%.

The bagasse feed, a high quality feed having sufficient digestibility,good taste and excellent in preservation, is suitable for long-distancetransportation.

Example B-6

One hundred parts by weight of an alkali-treated bagasse obtained by themethod in Example A-3 was blended uniformly with as nutrients 10 partsby weight of wheat flour bran, 10 parts by weight of de-fatted soybean,2 parts by weight of cottonseeds, 5 parts by weight of molasses, 1 partby weight of lactic acid and 40 parts by weight of water, and then thebagasse feed was obtained.

The bagasse feed, a high quality feed having sufficient digestibilityand good taste, is suitable for ruminants.

Example B-7

Twenty cattle having a body weight of 400 to 500 kg were fed for aperiod of 60 days by providing them with the fermented bagasse feedobtained by the method in Example B-3. The taste of the fermentedbagasse feed is very preferable for the cattle and their average intakeof the fermented bagasse feed in a day was about 20 to 25 kg/body, andfurther they increased their body weights by about 2.1 kg/body in a dayon an average in satisfactory physical and feeding conditions.

As described above, bagasse, an unutilized agricultural waste, isutilized according to the invention to prepare an alkali-treated bagassewithout decomposing cellulose and hemicellulose as an effectivecomponent by using as alkaline reagents calcium oxide together with orwithout sodium hydroxide, and further a good taste and high qualityfermented bagasse feed is produced easily within a relatively-shortperiod of time by inoculating lactic acid bacteria to the alkali-treatedbagasse and succeedingly fermenting the mixture.

It was found that the alkali-treated bagasse thus obtained and a bagassefeed as well as a fermented bagasse feed is usable as a feed forlivestock as well as advantageously utilizable as an organic fertilizer.

Accordingly, the present invention saves sugar-refinery industries fromthe difficulties of treating bagasse deemed as an unutilized industrialwaste and livestock farming industries from the shortage of roughage,and further is extremely significant in agriculture, industries ofsweetening products, feed industries and livestock product processors.Furthermore, in the case of standing in global view of observing theearth overall, it is no exaggeration to say that the present inventionestablished a novel technology to save our future facing anenvironmental disruption, an overflowing population and a food crisis byproviding a vast amount of foodstuff like livestock and milk productsfrom bagasse or unutilized biomass exhausted largely and annually,without any competition with our foods.

While there has been described what is at present considered to be thepreferred embodiments of the invention, it will be understood thevarious modifications may be made therein, and it is intended to coverin the appended claims all such modifications as fall within the truespirits and scope of the invention.

We claim:
 1. A biologically pure Enterococcus faecium FERM BP-4504,which is a bacterium capable of proliferating in an alkaline nutrientculture medium having a pH not less than 9.5.